Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that is used to recruit essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 recombinant human MIT domains. We measured 228 pairwise interactions, quantified 60 positive interactions, and discovered 18 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: (1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), (2) function in abscission (SPASTIN, KATNA1, and CAPN7), and (3) function in the ‘NoCut’ abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.


Introduction
To complete cell division, a cell must equally and fully partition a faithfully duplicated genome into two nascent progeny cells through mitosis, and these cells must physically separate through cytokinesis. In animal cells, cytokinesis initiates when an actomyosin contractile ring ingresses to create a cleavage furrow between the two spindle poles following chromosome segregation (Addi et al., 2018). The plasma membrane furrow ultimately narrows into an intercellular membrane bridge containing a dense central array of microtubules (the midbody), which then undergoes abscission to create two new cells. Premature abscission in the presence of aberrantly segregated DNA can lead to tensioninduced double strand breaks in chromatin bridges that traverse the cleavage furrow (Janssen et al., 2011), chromosome instability (Umbreit et al., 2020), and formation of micronuclei and extensive DNA damage through chromothripsis (Zhang et al., 2015;Crasta et al., 2012). Hence, abscission is required for cell proliferation and its misregulation can contribute to DNA damage and even cancer development (Pharoah et al., 2013;Sadler et al., 2018).
In addition to constricting the midbody, ESCRT-III filaments also recruit cofactors that contribute to abscission and checkpoint regulation. Human ESCRT-III proteins have conserved helical core domains that mediate filament formation Pfitzner et al., 2021;Bajorek et al., 2009b), and variable C-terminal tails that contain short peptide elements called MIT-Interacting Motifs (MIMs) ( Figure 1A). MIMs bind cofactors that contain MIT (Microtubule-Interacting and Trafficking) domains Figure 1B). MIT domains are simple three helix bundles, but they can bind MIM elements in at least seven distinct ways Obita et al., 2007;Scott et al., 2005;Stuchell-Brereton et al., 2007;Solomons et al., 2011;Fujioka et al., 2014), which we have termed Type 1-7 binding modes ( Figure 1C).
Humans express more than 20 MIT domain-containing proteins, which also contain a variety of associated activities that could function in cytokinesis ( Figure 1B

MIT-MIM interaction screen
We defined the ESCRT-III-MIT interactome by using fluorescence polarization anisotropy (FP) to quantify the binding interactions between recombinant human MIT domains and ESCRT-III MIM peptides. These experiments employed fluorescently labeled C-terminal peptides (52-88 residues) that encompass the known MIM elements of the 12 human ESCRT-III proteins ( Figure 1A, Supplementary file 1B). The ESCRT-III tails were fluorescently labeled at their N-termini. Literature analyses were used to identify 21 candidate human MIT domains (Ciccarelli et al., 2003;Rigden et al., 2009;Skalicky et al., 2012;Xiao et al., 2008;Fujioka et al., 2014; Figure 1B; see Materials and methods), which were expressed as recombinant proteins and purified to homogeneity. Constructs encoding the tandem MIT domains of LIP5, CAPN7, and ULK3 spanned both domains, thereby allowing intramolecular interactions to occur and native structures to form. Vild et al., 2015;Guo and Xu, 2015). 19/21 of the human MIT domain constructs could be purified in sufficient quantities for FP binding titrations (Figure 2). The two exceptions were the C-terminal tandem MIT domains of ULK1  (green). For clarity, the three helix bundles of the different MIT domains are shown in approximately equivalent orientations. Type 1 interaction: a helical MIM binds in the groove between MIT helices 2 and 3 (H2/H3 groove), and is oriented parallel to MIT helix 3. Type 2 interaction: the MIM forms an extended strand that binds in the MIT H1/H3 groove, parallel to MIT helix 3. Type 3 interaction: a helical MIM binds in the MIT H1/3 groove, parallel to MIT helix 3. Type 4 interaction: Similar to Type 1, except that the longer MIM helix binds lower in the H2/H3 groove and interacts with the H2/H3 loop. Type 5 interaction: Two MIM helices and adjacent linkers wrap nearly completely around the MIT domain. Type 6 interaction: a mixed helix/strand MIM element binds in the MIT H1/3 groove, antiparallel to MIT helix 3. Type 7 interaction: a helical MIM element binds in the MIT 2/3 groove, antiparallel to helix 3. The specific complexes shown in the figure are labeled below, together with their PDB accession codes. and ULK2, which function in autophagy (Fujioka et al., 2014) and apparently do not bind ESCRT-III proteins (see below). Pairwise FP binding isotherms were fit to 1:1 equilibrium binding models ( Figure 2, Figure 2figure supplements 1-4, Figure 3). Dissociation constants (K D ) for the 228 interactions tested are summarized in Figure 2 and color-coded based upon interaction strengths. Our screen generally recapitulated binding interactions reported previously using orthogonal techniques (See Supplemental         Discussion), but we cannot rule out the possibility that in some cases the position of the label could have interfered with or artificially enhanced binding interactions for some ESCRT-III-MIT pairs. Pairwise binding isotherms for weak binding pairs often did not reach saturation ( Figure 2-figure supplements 1-3), and the K D s for those interactions should therefore be considered approximations. In cases where isotherms did not reach half saturation at the highest MIT concentration tested (usually 100 µM), we did not attempt to estimate the K D .
Our binding survey confirmed a series of previously reported ESCRT-III-MIT domain interactions (See Supplemental discussion), and also revealed 18 previously unreported interactions (Figure 2), including establishing new direct links between ESCRT-III proteins and the MIT domains from KATNA1, KATNAL1, RPS6KC1, RPS6KL1, SNX15, and AMSHLP. These new interactions should facilitate future investigations of ESCRT-associated activities.  The seven promiscuous binders bound ESCRT-III tails with high (K D <2 µM) or moderate (K D <100 µM) affinities. The MIT domain of the deubiquitinase AMSH exhibited the greatest promiscuity, binding all but two of the 12 different ESCRT-III subunits. The eight specific binders interacted with CHMP3 (KATNA1, KATNAL1, RPS6KL1, RPS6KC1, and AMSHLP), IST1 (RPS6KC1, CAPN7, SNX15, and SPARTIN), or CHMP1B, CHMP3, and IST1 (SPASTIN). The predicted MIT domain of the catalytically inactive deubiquitinase, USP54 , bound weakly (K D >100 µM) to nearly all ESCRT-III proteins, indicating that these interactions were probably non-specific ( Figure 2-figure supplement 3). Finally, two MIT domains (NRBF2 and VPS9D1) did not bind any ESCRT-III proteins ( Figure 2-figure supplement 4). The MIT domain of NRBF2 has been characterized structurally (PDB 4ZEY, 2CRB) and shown to interact with the VPS15 kinase to promote autophagosome biogenesis (Young et al., 2019). The putative VPS9D1 MIT domain was predicted through bioinformatics, has not been characterized structurally, and has no known ESCRT connections ). The tightest MIM-MIT binding pair was LIP5-CHMP5 (K D = 2.5 nM), which reflects the fact that CHMP5 tail helices 5 and 6 and adjacent linkers form an amphipathic 'leucine collar' that wraps almost completely around the second LIP5 MIT domain ( Figure 1C; Skalicky et al., 2012).
At the other end of the spectrum, a fluorescently labeled CHMP4C peptide did not bind detectably to any MIT domain tested. This was surprising, particularly as MITD1, USP8, and AMSH all bound the other two CHMP4 paralogs, CHMP4A and CHMP4B ( Figure 2). CHMP4C is unique in containing a serine-rich insertion that is phosphorylated by AurB (Carlton et al., 2012;Capalbo et al., 2012;Figure 2-figure supplement 5A). We reasoned that this flexible insert might reduce the fluorescence anisotropy in bound complexes, thereby generating false negative results. To test this idea, we assayed whether the CHMP4C peptide could competitively inhibit AMSH, MITD1 and USP8 MIT binding to labeled CHMP4B (Figure 2-figure supplement 5B). These experiments revealed that CHMP4C bound AMSH MIT (K i 14 µM), but still failed to bind MITD1 or USP8. CHMP4C performs a unique role in abscission checkpoint regulation (Carlton et al., 2012;Capalbo et al., 2012), and this specialized function may have relieved the selective pressure to maintain some MIT binding interactions.
As noted above, we were unable to express and purify the ULK1 or ULK2 tandem MIT domains in sufficient quantities for direct binding assays. Nevertheless, we felt it was important to test whether the MIT domains from these related kinases could bind ESCRT-III proteins because: (1) the MIT domains of yeast and human ULK1/2 kinases bind ATG13 within autophagosome initiation complexes (Chan et al., 2009;Fujioka et al., 2014), and (2) the MIT domain of the related ULK3 kinase binds tightly to IST1, and weakly to CHMP1A, CHMP1B, CHMP2A, and CHMP3  and ( Figure 2). We therefore performed pulldown assays from human HEK239T cell extracts to test whether ULK1(MIT) 2 could bind ESCRT-III proteins (Figure 2-figure supplement 6A and B). Positive control pulldown assays recapitulated the known ULK3(MIT) 2 -ESCRT-III interactions, as well as ULK1(MIT) 2 -ATG13 binding. However, ULK1(MIT) 2 did not bind detectably to any of the ESCRT-III binding partners of ULK3, implying that these ESCRT-III interactions are not conserved in the ULK family of protein kinases.
In summary, most MIT proteins can engage ESCRT-III proteins and their interactions span a range of specificities and affinities, indicating that they couple to ESCRT pathway functions in a variety of different ways.

Structure and analysis of the SPASTIN MIT-IST1 complex
The SPASTIN MIT domain is unusual in binding specifically to three different ESCRT-III proteins: CHMP1B, CHMP3, and IST1. The SPASTIN MIT-CHMP1B complex has been characterized structurally  Figure  (1) The longer CHMP1B helix extends three additional turns beyond the IST1 N-terminus, and (2) the MIT H1/H3 groove expands to accommodate the longer CHMP1B helix, with a maximal displacement of 2.5 Å at the N-terminus of helix 3. The structure of SPASTIN-IST1 is nearly identical to SPARTIN-IST1, which also adopts a Type 3 interaction Figure 4-figure supplement 1D). IST1 binding is slightly tighter than CHMP1B binding, likely owing to enhanced hydrophobic interactions with the SPASTIN H1/ H3 groove (Figure 4-figure supplement 1C). IST1 buries two aromatic and two aliphatic side chains, whereas the CHMP1B binding element lacks aromatic residues and buries only three aliphatics.
Based on our structural analyses, we created SPASTIN MIT mutations designed to disrupt the binding of both CHMP1B and IST1 (F124D, red in Figure 4A-D) and to disrupt only CHMP1B binding without affecting IST1 binding (L177D, cyan in Figure 4A -D). As shown in Figure 4E and F, these mutations behaved as designed, thereby providing a set of mutants that we could use to compare the biological effects of disrupting SPASTIN binding to both CHMP1B and IST1 vs. specifically disrupting binding to CHMP1B alone. CHMP3 binding was also inhibited by both SPASTIN F124D and L177D mutants. Thus, CHMP3 also likely binds as an extended Type 3 helix in the SPASTIN MIT H1/H3 groove ( Figure 4-figure supplement 2), and only IST1 retained binding to the SPASTIN L177D mutant owing to its shorter Type 3 helix.

KATNA1 and CAPN7 binding to ESCRT-III proteins
We also screened for MIT point mutations that could block ESCRT-III binding to KATNA1 and CAPN7 by making a series of disruptive mutations in the MIT H1/H3 and H2/H3 grooves and testing whether these mutations abrogated binding in our fluorescence polarization anisotropy binding assay. In the KATNA1-CHMP3 case, a point mutation in the MIT H1/H3 groove (V55D) eliminated CHMP3 binding ( Figure 4G). Our data therefore again indicate a Type 3 interaction for the helical CHMP3 MIM element. CAPN7 has tandem MIT domains, and we found that IST1 binding was significantly reduced (~30-70 fold) by point mutations in the H1/H3 groove of either the first (V18D) or second (F98D) CAPN7 MIT domains ( Figure 4H). Together with our MIM mapping experiments ( Figure 3C), these data indicate that both CAPN7 MIT domains engage both IST1 MIM elements, employing Type 2 (MIM 316-343 ) and Type 3 (MIM 344-366 ) binding modes.

SPASTIN, KATNA1, and CAPN7 localize to midbodies
As an initial screen for cytokinetic functions, we examined whether endogenous SPASTIN, KATNA1, and/or CAPN7 localize to the midbodies of dividing cells ( Figure 5A-C, respectively). Midbodies were co-stained for microtubules and CEP55, which is recruited to either side of the Flemming body where it initiates ESCRT complex assembly (Fabbro et al., 2005;Carlton and Martin-Serrano, 2007;Morita et al., 2007). Antibody staining specificity was confirmed in parallel experiments in which each MIT protein was separately depleted using siRNA (Figure 5A-C; right panels).
In midbody-containing cells, all three MIT proteins co-localized with CEP55 ring structures on either side of the Flemming body ( Figure 5A-C). Uniquely, KATNA1 also distributed along the midbody arms, reminiscent of localization observed for caveolae, and termed 'midbody entry points' (Andrade et al., 2022). Arm staining was particularly prominent in early midbody-stage cells ( Figure 5B), and a similar KATNA1 staining pattern has been reported in rat cell lines (Matsuo et al., 2013). Our observations of SPASTIN and KATNA1 Flemming body localization are also in good agreement with previous reports Connell et al., 2009;Matsuo et al., 2013). Importantly ours is the first report that CAPN7 localizes to midbodies.
MIT protein localization was also examined in cells in which abscission checkpoint signaling was sustained by nucleoporin depletion (Strohacker et al., 2021;Figure 5D-F). In these experiments, cells were synchronized using thymidine treatment and release, together with siRNA depletion of NUP153 and NUP50 (siNups), which sustains the abscission checkpoint. In all cases, the MIT proteins again localized to Flemming bodies, although in each case abscission checkpoint signaling led to more diffuse staining and spreading of the signals to midbody arms. Thus SPASTIN, KATNA1, and CAPN7 localize to midbodies under both unperturbed and checkpoint active conditions, where they are poised to function in abscission and/or the abscission checkpoint.

SPASTIN, KATNA1, and CAPN7 function in abscission
To test for abscission functions, we depleted SPASTIN, KATNA1, and CAPN7 and quantified abscission failure, as reflected by increased numbers of multi-nucleated cells and cells with persistent intercellular bridges ( Figure 5G and Figure 5-figure supplement 1). Specific depletion and abscission phenotypes were confirmed using two different siRNA oligonucleotides in each case, and successful target protein depletion was confirmed by Western blot (Figure 5-figure supplement 1).
Cells lacking KATNA1 and CAPN7 both showed strong abscission failure phenotypes, with near doubling of the percentages of cells with midbodies or multiple nuclei (vs. control cells transfected with non-targeting (NT) siRNA). These pronounced phenotypes resembled the abscission defects observed with moderate knockdown of the essential IST1 protein (positive control) . Cells lacking SPASTIN also exhibited significant abscission defects, although the effects were weaker. Our results are consistent with previously reported roles for KATNA1   and SPASTIN in promoting abscission by clearing midbody spindle microtubules from abscission sites (Matsuo et al., 2013;Yang et al., 2008;Connell et al., 2009). The observation that CAPN7 is required for efficient cytokinetic abscission is a new discovery.

SPASTIN and CAPN7 are required for abscission checkpoint maintenance
We also tested whether SPASTIN, KATNA1, or CAPN7 were required to maintain the AurB-mediated abscission checkpoint. As described above, co-depletion of the nuclear pore proteins NUP153 and NUP50 sustains abscission checkpoint activity, leading to an accumulation of midbody-connected cells (Mackay et al., 2010). However, simultaneous co-depletion of either SPASTIN or CAPN7 significantly reduced midbody accumulation, indicating roles for both proteins in abscission checkpoint maintenance ( Figure 5H, Figure 5-figure supplement 1). Indeed, the effects of depleting either SPASTIN or CAPN7 were at least as penetrant as depleting CHMP4C (positive control), which plays a well-characterized role in inhibiting abscission in response to checkpoint signaling (Capalbo et al., 2012;Carlton et al., 2012). Unlike SPASTIN or CAPN7 depletion, KATNA1 depletion did not significantly alter midbody numbers, although a second oligo targeting KATNA1 showed a modest but significant decrease in cells undergoing an abscission checkpoint arrest ( Figure 5H, Figure 5-figure supplement 1). Thus, our data indicate that SPASTIN and CAPN7 are required to sustain abscission checkpoint arrest, and KATNA1 may also contribute weakly.

ESCRT-III proteins recruit SPASTIN, KATNA1, and CAPN7 to midbodies
To unite our binding and functional data, we tested whether ESCRT-III interactions were responsible for recruiting SPASTIN, KATNA1 and CAPN7 to function at midbodies. This was done by generating cell lines that expressed doxycycline (DOX)-inducible, siRNA-resistant mCherry constructs fused to wildtype (WT) and mutant MIT proteins. We then treated with siRNA to deplete the respective endogenous MIT proteins (Figure 6-figure supplement 1) while inducing expression of the mCherry fusion proteins and imaging the cells to test for midbody localization ( Figure 6). As in Figure 5, cells were also synchronized with sustained checkpoints to maximize the number of midbody-stage cells (Strohacker et al., 2021). Importantly, all three WT mCherry fusion constructs recapitulated localization of the endogenous MIT counterpart within the midbody ( Figure 6). The percentages of "arms only" localization patterns were higher for the endogenous constructs in every case, however, potentially reflecting reduced antibody epitope accessibility at the protein-rich Flemming body. We found some additional differences between expression constructs and their individual endogenous counterparts, such as a larger population of "Flemming Body + Arms" in mCherry-SPASTIN cells, which again may reflect epitope exposure ( Figure 6A). These observations suggest that there may be more variation in SPASTIN localization than currently appreciated, particularly when the abscission checkpoint is sustained. For purposes here, however, this assay provides a robust system in which to probe the requirements for ESCRT-III-mediated recruitment to the midbody.
Like WT mCherry-SPASTIN, the L177D mutant SPASTIN localized to more than half of all midbodies ( Figure 6A and B). The proportion of midbodies occupied by SPASTIN may reflect a temporally restricted role and/or be influenced by overall sensitivity of the assay. Strikingly, however, the F124D mutant did not localize to midbodies. These data imply that ESCRT-III binding is required to localize SPASTIN to the midbody. The SPASTIN F124D mutation disrupts binding to CHMP1B, IST1, and CHMP3 whereas the L177D mutation selectively permits only IST1 binding (Figure 4, Figure 4-figure selected regions are shown at the right of each image, with the target protein shown alone (top) or together with CEP55 (bottom). (G, H) Quantification of abscission defects (midbody-stage cells and multinucleate cells) under asynchronous conditions (G) or with a sustained abscission checkpoint (H). Bars represent the average and standard deviation from n=5 independent experiments where N>500 cells were counted per experiment. Statistical analysis was performed using ANOVA, comparing total abscission defects (Midbody-stage cells + Multinucleate cells) after each individual siRNA treatment to siNT. ****p<0.0001, ***p<0.0005, **p<0.02, ns = not significant.
The online version of this article includes the following source data and figure supplement(s) for figure 5:    supplement 2). Therefore, our data imply that CHMP1B and CHMP3 binding are dispensable for midbody localization when IST1 binding capability is maintained.
The KATNA1 MIT domain binds the C-terminal domain of the KATNB1 subunit to form the KATNA1 holoenzyme (Faltova et al., 2019;Rezabkova et al., 2017). KATNB1 activates and localizes KATNA1 to centrosomes where it severs mitotic spindle microtubules (Matsuo et al., 2013). However, KATNB1 does not appear to be responsible for localizing KATNA1 for cytokinesis function, as the two proteins reportedly do not co-localize within the midbody (Matsuo et al., 2013). It has therefore not been clear how KATNA1 is recruited to the midbody to function in abscission. Our localization data demonstrate that WT mCherry-KATNA1 localizes to Flemming bodies and midbody arms, but the V55D mutant does not ( Figure 6C and D). CHMP3 is the sole ESCRT-III binding partner of KATNA1, and the V55D mutation disrupts this partnership (Figure 3), implying that CHMP3 localizes KATNA1 to the midbody for abscission.
The structure of the KATNA1 MIT-KATNB1 heterodimer reveals that V55 sits adjacent to the KATNB1 binding site Figure 6- figure supplement 2A), and we therefore tested the possibility that the V55D mutation might also disrupt the canonical KATNA1-KATNB1 partnership. This was not the case, however, as KATNB1 co-precipitated with full length KATNA1 WT and V55D mutant proteins equally well from HEK293T cell extracts ( Figure 6-figure supplement 2B). In contrast, a KATNA1 MIT mutation known to disrupt KATNB1 binding, R14A , abolished KATNB1 binding, demonstrating that the pulldown assay was sensitive to specific disruption. Hence, our data imply that the KATNA1 V55D mutation discriminates between CHMP3 and KATNB1 binding, and therefore that CHMP3 is likely responsible for localizing KATNA1 to Flemming bodies. Consistent with this model, CHMP3 localizes to Flemming bodies (Mierzwa et al., 2017;Dukes et al., 2008), and its depletion increases the propensity of cells to develop multiple nuclei and midbody bridges (Morita et al., 2010), thereby phenocopying KATNA1 loss ( Figure 5G).
Finally, our MIT screening data suggested that IST1 could be responsible for localizing CAPN7 to midbodies (Figure 2). In support of this model, a point mutation in the second CAPN7 MIT domain that decreased IST1 binding ~30-fold (F98D, see Figure 3) also potently suppressed CAPN7 targeting to the Flemming body ( Figure 6E and F). Thus, in all three cases we examined in detail, our MIT screening data identified potential ESCRT-III binding partners and point mutations that blocked binding, and in every case the point mutations also abolished MIT protein midbody localization.

Discussion
We have comprehensively surveyed the MIT-ESCRT-III interactions between human ESCRT-III and MIT domain-containing proteins. We tested 228 pairwise interactions between MIT domains and ESCRT-III C-terminal tails, observed 60 positive interactions, and discovered 18 new interactions (Figures 2  and 3, see Supplemental discussion). We found that most human MIT domains can mediate ESCRT-III binding, further implicating MIT proteins as a major class of ESCRT pathway cofactors. SPASTIN, KATNA1, and CAPN7 served as paradigms for using the binding data to discover new ESCRT cofactors that function in cytokinesis. Our screening assays identified ESCRT-III binding partners and interaction sites (Figure 3), enabled new structure determinations (Figure 4, Figure 4-figure supplement 1), and supported mutational analyses of MIT protein midbody localization (Figure 4, except that endogenous KATNA1 was depleted by siRNA in the DOX-inducible cell lines. Statistical analysis was performed using an unpaired t-test, comparing the V55D mutant to WT. (E, F) Immunofluorescence and quantification of midbody localization for endogenous CAPN7 ( Figure 5) and DOXinducible cell lines expressing siRNA-resistant CAPN7-mCherry and CAPN7-F98D-mCherry constructs as in A and B, except that endogenous CAPN7 was depleted by siRNA in the DOX-inducible cell lines. Statistical analysis was performed using an unpaired t-test comparing the F98D mutant to WT. ****p<0.0001, ***p=0.0005, **p=0.0024, ns = not significant.
The online version of this article includes the following source data and figure supplement(s) for figure 6:      Figure 6). Together with previous studies, our work indicates that the primary biological function of MIT domains is to localize proteins to sites of ESCRT-driven membrane remodeling. MIT domains have also evolved to mediate protein-protein interactions in other pathways, most notably in autophagosome initiation (Fujioka et al., 2014;Young et al., 2019). Collectively, MIT-MIM interactions constitute a complex recognition network, and several principles that emerged from our studies are highlighted below. A more comprehensive discussion of the different interactions is provided in the Supplemental discussion.

MIT recruitment by ESCRT-III polymers
Although we studied the pairwise interactions of MIT domains and ESCRT-III tails, these interactions likely occur predominantly in the context of polymeric ESCRT-III filaments. Polymerization creates the potential for high avidity binding, and variations in ESCRT-III subunit compositions can alter binding modes and tune binding affinities. These properties are important because subunit compositions change as filaments mature and constrict membranes (Pfitzner et al., 2021;Banjade et al., 2021;Pfitzner et al., 2020). Current models for ESCRT-III assembly, derived from studies in simpler yeast systems, hold that CHMP6 initially nucleates the assembly of ESCRT-III strands composed of CHMP4 subunits. CHMP4 strands then recruit partner strands composed of CHMP2/CHMP3 subunits (Teis et al., 2008;Babst et al., 2002), which are subsequently exchanged for CHMP1/IST1 subunits . Hence, the cadre of recruited MIT protein binding partners may evolve as filaments mature.
These different principles are nicely illustrated by VPS4 enzymes and their associated activator LIP5 (Vta1 in yeast), which form 'supercomplexes' that display 30 MIT domains; six from the VPS4 hexamer and 24 from the 12 associated LIP5(MIT) 2 elements Yang et al., 2012). VPS4 MIT domains bind promiscuously to many ESCRT-III tails, and although binding is often weak avidity effects can presumably create high affinity binding. We measured strong to moderate, Type 1 VPS4 MIT binding to the late-acting CHMP1, CHMP2 and IST1 subunits (nM to mid-μM K D s), with three-to 20-fold higher affinities consistently seen for VPS4A MIT vs. VPS4B MIT. CHMP3 binding was below our 200 μM binding affinity cutoff for both VPS4 MIT domains, and the only case in which weaker, earlyacting Type 2 interactors CHMP6 and CHMP4 made our binding cutoff was in the VPS4A MIT-CHMP6 complex. Nevertheless, there is good evidence that interactions between CHMP4 (Snf7 in yeast) and VPS4 are biologically important Shestakova et al., 2010;Buysse et al., 2020). The first of the tandem LIP5 MIT domains similarly binds ESCRT-III tails promiscuously, and again favors late-acting ESCRT-III subunits , whereas the second LIP5 MIT domain binds specifically and with high affinity to CHMP5 Yang et al., 2012). Thus, these MIT interactions can collectively explain how (VPS4) 6 (LIP5) 12 supercomplexes can bind and remodel ESCRT-III filaments that contain essentially any combination of the 12 different human ESCRT-III subunits. Moreover, their different binding affinities can explain why VPS4 remodeling activity increases as ESCRT-III filaments mature and accumulate increasing fractions of the late-acting, and higher affinity CHMP2, CHMP1, and IST1 subunits .
In a similar fashion, ESCRT-III subunit binding specificities and affinities are also likely tuned to recruit other MIT cofactors as needed. It is therefore of interest that IST1, CHMP3, and CHMP1 were the most promiscuous ESCRT-III binders in our screen because each of these ESCRT-III subunits functions at transitional stages of ESCRT-III polymer maturation . Our binding data suggest that many MIT proteins will bind best to mature, IST1-containing filaments that form late, when membrane constriction is greatest and fission is most imminent. In contrast, MIT proteins like AMSH and MITD1 bind many different ESCRT-III subunits with similar affinities, perhaps because deubiquitination (AMSH) and midbody stabilization (MITD1) are required throughout abscission and other ESCRT-dependent processes.

IST1 as a versatile hub for cytokinetic cofactor recruitment
IST1 is the most promiscuous MIT binding ESCRT-III protein, and it binds most targets with high affinity (Figure 2, Figure 3C). IST1 promiscuity is achieved through combined use of two different MIM elements, and through multi-modal binding by the IST1 MIM1/3 element (Bajorek et al., 2009a, Guo and Figure 3). Our screen revealed that both IST1 MIM elements can mediate MITbinding, and that they often collaborate to enhance binding ( Figure 3C). The SPASTIN MIT-IST1 structure shows that the IST1 MIM 344-366 element can make Type 3 MIT interactions, as seen previously in the SPARTIN-MIT-IST1 complex Guo and Xu, 2015). However, the same hydrophobic surface of the IST1 MIM 344-366 helix can also form Type 1 interactions with the MIT domains of ULK3, VPS4B, and LIP5 Skalicky et al., 2012;Caballe et al., 2015). IST1 depletion induces severe cytokinetic defects Figure 5), reflecting its central role in abscission and as a versatile hub for MIT cofactor recruitment.
Comparison of the SPASTIN MIT complexes with IST1 and CHMP1B allowed us to design a mutation that specifically permitted only IST1 binding (Figure 4). This SPASTIN mutant still localized to intercellular bridges ( Figure 6A,B) demonstrating that IST1 binding is sufficient (and CHMP1B and CHMP3 are dispensable) for midbody recruitment. Others have reported that SPASTIN midbody localization is impaired by CHMP1B depletion , but this observation can be reconciled because IST1 recruitment is also likely affected by the absence of CHMP1B (Dimaano et al., 2008;Rue et al., 2008;Bajorek et al., 2009b, Goliand et al., 2018. MIT binding modes of CHMP1 and CHMP3 proteins CHMP1B and CHMP3 are also promiscuous MIT binders. CHMP1B binds two MIT domains (SPASTIN, USP8) that the closely related CHMP1A protein does not. This expanded binding range apparently reflects the ability of CHMP1B to access the Type 3 interaction mode. The Type 3 interaction extends the N-terminus of the CHMP1B MIM helix by three additional turns (vs. its Type 1 interactions). Additional interactions with this extended helix could allow SPASTIN to discriminate between CHMP1B and CHMP1A, whose sequences are highly similar at the C-terminus but diverge N-terminally . The USP8 MIT domain can also discriminate between CHMP1B and CHMP1A (Figure 2), and we therefore speculate that USP8 MIT may also form a Type 3 interaction with CHMP1B.
The CHMP3 MIM appears to be another example of a promiscuous helical MIM element that can bind in at least two separate grooves of different MIT domains. The CHMP3 MIM element binds the MIT domains of AMSH using a Type 4 mode ( Figure 1C; Solomons et al., 2011), and LIP5 (MIT1 using a Type1/4 mode; Skalicky et al., 2012). Remarkably, our study suggests that CHMP3 can also adopt yet another binding mode (Type 3), as we mapped binding to the H1/H3 grooves of SPASTIN and KATNA1 MIT. The ability to adopt multiple different binding modes increases the number of possible partnerships with MIT domain proteins, and helps to explain why IST1, CHMP3 and CHMP1B are the most promiscuous MIT binders.

SPASTIN and KATNA1 midbody recruitment and microtubule severing
ESCRT-III proteins coordinate the recruitment of the microtubule severing enzymes KATNA1 and SPASTIN to complete abscission. Following anaphase, densely packed midbody microtubules must be cleared from the midbody to facilitate cytokinetic membrane abscission (Sharp and Ross, 2012). Both SPASTIN and KATNA1 form hexameric rings (Hartman and Vale, 1999;Eckert et al., 2012) that can engage the C-terminal tails of TUBULIN subunits within the central pore (Kuo and Howard, 2021). ATP hydrolysis then drives polypeptide translocation, thereby promoting subunit exchange and/or microtubule severing (Roll-Mecak and Vale, 2008;Zehr et al., 2017). We observed distinct but overlapping midbody localization patterns for SPASTIN and KATNA1 (Figure 5), which may reflect their recruitment by similar ESCRT-III proteins (CHMP1B, IST1, and CHMP3) and could allow the two enzymes to act on different pools of midbody microtubules.
Our discovery that SPASTIN, but not necessarily KATNA1, is required for abscission checkpoint maintenance further underscores that these enzymes likely mediate different aspects of microtubule dynamics during abscission. Furthermore, the surprising checkpoint requirement for SPASTIN suggests that microtubule severing may also be required to support abscission arrest (in addition to physically removing microtubule barriers to allow abscission). Possible roles for SPASTIN in maintaining abscission arrest include: (1) slowing microtubule catastrophe via an ATP-independent activity (Kuo et al., 2019) and thereby paradoxically stabilizing severed midbody microtubules, or (2) creating shorter microtubules that promote regrowth and dynamically increase microtubule networks (Kuo et al., 2019;Vemu et al., 2018), and could thereby stabilize the midbody and/or traffic abscission factors into or out of the abscission zone (Frémont and Echard, 2018). Our findings point to the need for more detailed studies of the mechanistic roles of microtubule remodeling in abscission and in checkpoint regulation.
The discovery that CHMP3 and KATNB1  share overlapping binding sites on the KATNA1 MIT domain has important functional implications (Figure 6-figure supplement  2). KATNB1 targets KATNA1 to centrosomes during mitosis (Matsuo et al., 2013), whereas CHMP3 targets KATN1A to midbody arms during cytokinesis ( Figure 6). Thus, KATNA1 likely switches partnerships from KATNB1 to CHMP3 during this cell cycle transition. KATNB1 binding also regulates KATNA1 microtubule severing activities (Faltova et al., 2019), specificities (Faltova et al., 2019), and binding partners Jiang et al., 2018), and it will therefore be of interest to learn how CHMP3 replacement alters these KATNA1 activities.
Finally, a CHMP3 mutation, T173I, located within the MIM used in our binding studies, is associated with spastic paraplegia, a disease associated with SPASTIN defects (Cohen-Barak et al., 2022). Our identification of CHMP3 as a SPASTIN binding partner suggests that CHMP3 may play a direct role in supporting neuronal SPASTIN functions.

CAPN7 functions in abscission and the abscission checkpoint
Finally, we have discovered that the cysteine protease, CAPN7, localizes to midbodies and supports abscission and the abscission checkpoint. Our screen identified IST1 as the sole ESCRT-III binding partner for the CAPN7 MIT domains (Figure 2, Figure 3), in good agreement with previous reports of IST1 binding in pulldown assays (Osako et al., 2010;Maemoto et al., 2011). The CAPN7 MIT domain also reportedly binds the second alpha-helical region of the CHMP1B core domain (but not the C-terminal MIM region) (Maemoto et al., 2011). We did not survey this interaction because our screen included only C-terminal ESCRT-III tails, but our mutational and localization analyses indicate that CAPN7 midbody recruitment is dependent on IST1 binding (Figure 4, Figure 6).
IST1 recruitment may also enhance CAPN7 proteolytic activity within the midbody as IST1 binding was shown to activate CAPN7 proteolysis of an artificial substrate (Osako et al., 2010;Maemoto et al., 2013). CAPN7 orthologues in Aspergillus (PalB) and budding yeast (Rim13) function together with ESCRT-III binding partners to cleave the PEST peptide sequences of the transcription factors Rim101 and PacC, and thereby enhance gene expression (Rodríguez-Galán et al., 2009;Subramanian et al., 2012). Similarly, human CAPN7 reportedly binds and cleaves PEST elements of the HOXA10 transcription factor (Yan et al., 2018). Thus, IST1 recruitment of CAPN7 to the midbody could provide spatial and temporal control of proteolysis, leading to downstream signaling activities required for checkpoint maintenance and/or abscission.
Expression inserts for cell lines were generated by PCR amplification and ligated into the pLVXtight-Puro vector (Clontech) using the NEB HiFi DNA Assembly Kit (New England Biolabs) according to the manufacturer's instructions. Human SPASTIN has four major isoforms (Mancuso and Rugarli, 2008;Claudiani et al., 2005), and we used the most abundant (M87) isoform, including residues encoded by exon 4.
A slightly longer SPASTIN MIT construct (residues 108-200) bound five-fold more tightly to CHMP1B and IST1 (CHMP1B, K D = 2.3 µM; Figure 2) compared to the minimal MIT sequence (residues 112-196; CHMP1B K D ~12-15 µM; data not shown and Yang et al., 2008) thus we used the longer construct for binding measurements. Using this construct, we were able to detect binding between SPASTIN and CHMP3; a previously unknown interaction.
Gene names, DNA sources, and amino acid sequences are given in Supplementary file 1 and Supplementary file 2. All plasmids generated by this study have been deposited to Addgene for distribution (See Supplementary file 2A for Addgene accession numbers).

Bacterial expression of MIT domains
Proteins were expressed in BL21 RIPL cells grown in ZYP-5052 autoinduction media (Studier, 2005). Transformed cells were initially grown for 3-6 hr at 37 °C, and then switched to 19 °C for an additional 20 hr. Cells were harvested by centrifugation at 5,400 x g and cell pellets were stored at -80 °C. MIT domains from VPS4A, VPS4B, ULK3, and LIP5 were expressed and purified as described previously Skalicky et al., 2012;Scott et al., 2005;Stuchell-Brereton et al., 2007).
Purification protocols for the CAPN7, SNX15 and VSP9D1 MIT domains were sufficiently different to merit separate descriptions (below).
(His) 6 -GST-CAPN7(MIT) 2 was bound to GST-sepharose beads (10 mL,GE Healthcare Life Sciences, USA, 6 hr), washed with 1 L wash buffer: 25 mM Tris (pH 8), 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and eluted with ~50 mL of wash buffer supplemented with 20 mM reduced L-glutathione (pH 8). The fusion tag was removed using ~100 µg GST-HRV3C (preScission) protease in a 3.5 kDa cutoff dialysis bag while dialyzing against 2x2 L wash buffer and 2 L of wash buffer with low salt (50 mM NaCl) over two days. The dialysate was purified by Q Sepharose chromatography (50 mL; GE Healthcare Life Sciences, USA) with a linear gradient of 50-500 mM NaCl. Fractions containing highly pure CAPN7(MIT) 2 were pooled and concentrated for gel filtration chromatography.
(His) 6 -GST-SNX15 MIT was bound to cOmplete His-Tag purification beads (5 mL, Roche, Germany, 2 hr) and washed with 2 L wash buffer: 25 mM Tris (pH 8), 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and eluted with wash buffer supplemented with 250 mM imidazole (pH 8). The eluent was bound to glutathione sepharose beads (10 mL, GE Healthcare Life Sciences, USA, 6 hr), washed with 1 L wash buffer: 25 mM Tris (pH 8), 500 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and eluted with ~50 mL of wash buffer supplemented with 20 mM reduced L-glutathione (pH 8). The fusion tag was removed using ~100 µg GST-HRV3C (preScission) protease in a 3.5 kDa cutoff dialysis bag while dialyzing against 2x2 L wash buffer and 2x2 L of wash buffer with low salt (25 mM NaCl) over 2 days. The dialysate was purified by Q Sepharose chromatography (50 mL, GE Healthcare Life Sciences, USA). SNX15 MIT eluted in the flow through and was concentrated for gel filtration chromatography.
(His) 6 -GST-VPS9D1 MIT was lysed in 50 mM Tris pH 7.4 (25 °C), 500 mM NaCl, 0.5 mM EDTA, and 1 mM TCEP supplemented with 0.125% sodium deoxycholate, lysozyme, PMSF, pepstatin, leupeptin, aprotinin and DNAse I (as described above). The clarified cell lysate was bound to 10 mL of cOmplete His-Tag purification resin (Roche, Germany, 30 min), washed with 1 L base buffer, and eluted with ~50 mL of base buffer supplemented with 250 mM imidazole (pH 8.0). The fusion tag was removed by incubation with ~100 µg GST-HRV3C protease in 3.5 kDa cutoff dialysis bag while dialyzing against 2x2 L of 25 mM Tris pH 7.4 (25 °C), 50 mM NaCl, 1 mM DTT, 0.5 mM EDTA. The dialysate was purified by Q Sepharose chromatography (50 mL, GE Healthcare Life Sciences, USA) and eluted with a 50-1000 mM NaCl linear gradient. VPS9D1 eluted in the flow through and was concentrated for gel filtration chromatography.
Finally, all MIT proteins were purified by Superdex 75 gel filtration chromatography (GE Healthcare Life Sciences, USA) in 25 mM Tris (pH 7.2 at 25 °C), 150 mM NaCl, 1 mM DTT, and 0.5 mM EDTA. MIT protein fractions were pooled and concentrated. MIT domain masses were confirmed with ESI-MS or MALDI-TOF (University of Utah Mass Spectrometry Core Facility, see Supplementary file 1A). Yields ranged between 0.5-35 mg/L of bacterial culture.

Expression and purification of ESCRT-III C-terminal peptides
ESCRT-III C-terminal peptides were prepared as described previously (Talledge et al., 2018). Briefly, most peptides were expressed as (His) 6 -SUMO-fusions, except for IST1-MIM 316-343 and IST1 MIM 344-366 which were made synthetically by the University of Utah Peptide Synthesis Core. Peptides were expressed in BL21-Codon Plus (DE3) RIPL cells (Agilent, Santa Clara, CA, USA) in ZYP-5052 autoinduction media (Studier, 2005). Cells were lysed by sonication in lysis buffer (40 mL/L of culture) containing 50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM imidazole, 2 mM DTT, 0.5 mM EDTA, and 0.125% sodium deoxycholate, supplemented with lysozyme, protease inhibitors, and DNAse I (Roche, Germany) (as described above). Clarified cell lysates were incubated with 10 mL of cOmplete His-Tag purification resin (Roche, Germany) for 30 min, washed with 500 mL wash buffer: 50 mM Tris, pH 7.2, 500 mM NaCl, 5 mM imidazole, 5 mM DTT, 0.5 mM EDTA, and then with 500 mL wash buffer containing 150 mM NaCl. (His) 6 -SUMO affinity tags were removed by on-column cleavage with (His) 6 -ULP1 (0.7 mg, overnight, 4 °C) in 40 mL of the 150 mM NaCl wash buffer. Cleaved peptides were collected from the column flow through and dialyzed against 25 mM sodium phosphate, pH 6.5, 50 mM NaCl, 2 mM DTT, 0.5 mM EDTA, and then purified by Q-sepharose ion exchange chromatography (GE Healthcare Life Sciences, USA) with a linear gradient from 50 mM to 1 M NaCl. Peptide fractions were pooled and dialyzed against 25 mM Tris, pH 7.2, 150 mM NaCl, 1 mM TCEP, 0.5 mM EDTA, and further purified by Superdex-75 size exclusion chromatography (GE Healthcare Life Sciences, USA). Typical IST1 peptide yields were 4.5 mg/L culture. Purified ESCRT-III C-terminal fragments contained non-native 'GlyCys' or 'Cys' residues at their N-termini, and masses were confirmed by mass spectrometry (see Supplementary file 1B) either before labeling (CHMP4C, or after labeling (all other peptides; dye adds a mass shift of 463.4 Da). Competition experiments used peptides lacking N-terminal 'Cys' residues to avoid disulfide cross-linking (see Supplementary file 1C).

Peptide fluorescent labeling
Fluorescent labeling was performed by the University of Utah DNA/Peptide Synthesis Core as described previously Talledge et al., 2018). Briefly, peptides were labeled in DMSO using ~1.3-fold molar excess of Oregon Green 488 maleimide (Life Technologies/Molecular Probes #O6034, USA) dissolved in a 1:1 solution of acetonitrile:DMSO. Reversed phase HPLC was used to monitor the reactions and separate labeled peptides from unreacted dye and unlabeled peptides using reversed phase HPLC. Labeled peptide fractions were dried under vacuum and dissolved in water. Peptide concentrations were quantified using the absorbance of Oregon Green 488 at 491 nm (e=83,000 cm −1 M −1 in 50 mM potassium phosphate, pH 9.0).

Fluorescence polarization anisotropy binding assays
Binding experiments were performed as described previously  in 25 mM Tris, pH 7.2, 150 mM NaCl, 0.1 mg/mL Bovine Serum Albumin (BSA), 0.01% Tween-20, and 1 mM DTT, with 250-500 pM fluor-labeled ESCRT-III peptides and purified MIT domains at the indicated concentrations. A Biotek Synergy Neo Multi-Mode plate reader (Biotek, USA) was used to measure fluorescence polarization with excitation at 485 nm and emission (detection) at 535 nm. Binding isotherms were fit to 1:1 models using KaleidaGraph (Synergy Software) as described previously Talledge et al., 2018). Reported K D values are averages from at least three independent isotherms. Non-binding (K D >200 µM) was confirmed at least twice independently. The interactions between IST1 and SPASTIN as well as IST1 and USP8 failed to generate the large changes in polarization needed to reliably fit dissociation constants. We therefore used a C-terminal labeled IST1 peptide to measure binding with SPASTIN, and competitive binding experiments were used to calculate the K i for the interaction of USP8 with IST1 ( Figure 2-figure supplement 5E). Competition experiments were performed as described previously (Sadler et al., 2018). Briefly, complexes of MIT domains and fluorescently labeled peptides (26.6 µM MITD1 and 0.5 nM CHMP4B peptide; 0.75 µM USP8 and 0.5 nM IST1 peptide, 12.5 µM USP8 MIT and 0.5 nM CHMP4B peptide) were titrated with the indicated concentrations of unlabeled peptides. IC 50 s were calculated using KaleidaGraph (Synergy Software) and then converted to K i values (Cer et al., 2009). Competitive binding curves were measured independently three or more times for each peptide, and one to three times for each positive control peptide.

Co-immunoprecipitation experiments KATNA1 pulldowns
HEK293T cells were seeded at 0.5x10 6 cells per well in six-well plates and transfected 24 hr later with 1 µg of plasmid encoding Myc-KATNB1 and one of the following: 2 µg empty vector control, 1.5 µg OSF-KATNA1-WT, 2 µg of OSF-KATNA1-R14A, or 1.5 µg of OSF-KATNA1-V55D P60 using PEI (10 ul per well of 1 mg/mL). Empty vector was added as necessary to bring the total transfected DNA to 3 µg/well. Cells were harvested 48 hr post-transfection and lysed in 400 µl of 50 mM Tris, pH 7.2, 150 mM NaCl, 0.5% Triton-X100, 1 mM DTT, protease inhibitors (1:100, Sigma). Lysates were clarified by centrifugation at 16,100 x g for 10 min at 4 °C, and clarified lysates were incubated with 20 µl of a 50% slurry of Strep-Tactin resin (IBA Biosciences) for 30 min. Beads were washed 4 x with 500 µl lysis buffer. After the final wash, Strep-Tactin beads were aspirated to near dryness and bound proteins were eluted by boiling in 40 µl of 2 x Laemmli sample buffer, resolved by SDS-PAGE, and detected by Western blotting.
The model was evaluated using Molprobity (Davis et al., 2007;Williams et al., 2018) and judged to be of good quality. Two regions of unexplained density were present in the Fo-Fc electron density map. PEG 300 is present at 40% (v/v) in the crystallization condition and the density resembled smaller PEG molecules. Several molecules were tested by model building and refinement and the best fit to the density was obtained with one molecule of triethylene glycol (PGE) and one of tetraethylene glycol (PG4). The PEG molecules improved the model statistics and maintained good geometry. Two initially assigned water molecules also produced unexplained density in the Fo-Fc electron density map. Several ions were tested by model building and refinement until the best fit was obtained with one chloride ion and one calcium ion. Both ions are present in the crystallization condition (hydrochloric acid and calcium acetate), and their inclusion improved the model statistics and maintained good geometry. The final model refined to R work = 0.149 and R free = 0.159. Structure coordinates have been deposited in the RCSB Protein Data Bank under PDB ID 7S7J. Full statistics and data collection details are provided in Supplementary file 3.
An omit map of the IST1 portion of the complex was generated for figure presentation. This map was calculated by removing IST1 from the final model followed with phenix refinement and simulated annealing (Cartesian) to minimize phase bias (Figure 4-figure supplement 1).

Cell culture
HEK293T and HeLa cells were cultured and maintained at 37 °C and 5% CO 2 in DMEM supplemented with 10% FBS. TetOn-HeLa cells were supplemented with 500 μg/mL G418 (Invitrogen) to maintain expression of the Tet-On Advanced protein. DOX-inducible cell lines generated in the parental TetOn-HeLa cell line were supplemented with 500 μg/mL G418 + 0.5 μg/mL puromycin (Invivogen).

Cell lines
Our parental HeLa cell line was authenticated by genomic sequencing of 24 loci (University of Utah Sequencing Core) and confirmed to be mycoplasma-free by routine PCR testing (ABM) following the manufacturer's protocols. HeLa cells were transfected with the pLVX-TetOn-Advanced plasmid (Clontech) and selected with 500 μg/mL G418 for 14 days. Single colonies were isolated, expanded, and screened for TetOn-Advanced expression by western blot using a TetR monoclonal antibody. The optimal clone, which was selected based on a combination of TetOn Advanced expression and tight control of DOX-inducible expression, was used as the parental HeLa TetOn line. To generate stable cell lines with doxycycline-inducible expression, the parental TetOn cell line was transfected with pLVX-tight puro plasmids containing the MIT genes of interest (see Supplementary file 2A) and selected for 14 days in 500 μg/mL G418 +0.5 μg/mL puromycin. Single colonies were expanded and screened for expression by immunofluorescence and western blotting. Selected clones were further validated by sequencing the PCR amplified MIT gene of interest from genomic DNA. Protein expression was induced by addition of 1 μg/mL doxycycline.

siRNA transfections
For experiments in Figure 5, transfection protocols were as follows: Day 1-350,000 cells were reverse transfected with 20 nM siRNA targeting MIT protein (as indicated) in a 35 mm dish using Lipofectamine RNAiMAX and following manufacturer's instructions; Day 2 -cells from Day 1 were trypsinized, resuspended in a total volume of 6 mL DMEM, and divided as follows: 0.5 mL into each of four wells of a 24-well dish containing 12 mm circle glass coverslips, and 2 mL into each of two 35 mm dishes. These samples were again reverse transfected with 20 nM siRNA (as indicated); for active abscission checkpoint samples, cells in two wells of a 24-well dish (for Immunofluorescence) and one 35 mm dish (for Western blot) were also reverse transfected with 10 nM siNUP153 and 10 nM siNUP50 at this time, incubated for 8 hr, then treated with 2 mM thymidine for 24 hr; Day 3 -thymidine was removed, cells were washed 2 x with warm PBS, and fresh medium added to all cells; Day 4-16 hr after thymidine release cells were harvested for analysis. For localization experiments in Figure 6, protein expression was induced by incubating cells in the presence of 1 μg/mL doxycycline for the duration of the 48 hr experiment. siRNA transfection protocols (in the presence of doxycycline) were as follows: Day 1 -either 70,000 cells (in one well of a 24-well dish containing a 12 mm glass coverslip for IF) or 350,000 cells (in a 35 mm dish for Western Blot) were reverse transfected with 20 nM siRNA targeting the indicated MIT protein plus 10 nM siNUP153 and 10 nM siNUP50 (siNups), incubated for 8 hr, then treated with 2 mM thymidine for 24 hr; Day 2 -thymidine was removed and fresh medium including 1 μg/mL doxycycline was added to all cells (as described above); Days 3-16 hr after thymidine release cells were harvested for analysis. siRNA sequences are reported in Supplementary file 2B.

Immunoblotting
Cells were lysed in RIPA buffer (Thermo Fisher) supplemented with mammalian protease inhibitor cocktail (Sigma; used at 1:100 dilution) for 15 min on ice with brief vortexing every 5 min. Lysates were cleared by centrifugation at 17,000 x g for 10 min at 4 °C. Lysate protein concentrations were determined using the BCA Assay (Thermo Fisher) and normalized prior to SDS-PAGE. 12 μg lysate per sample were prepared with SDS loading buffer, resolved by SDS-PAGE, and transferred to either PVDF or nitrocellulose. Membranes were blocked for 1 hr at room temperature in 5% milk in TBS, then incubated overnight at 4 °C with primary antibodies (see Supplementary file 2C for dilutions). Following 3x10 min washes in TBS-T, membranes were incubated with the corresponding secondary antibodies for 1 hr at 23 °C, washed again with TBS-T, and imaged using a LiCor Odyssey infrared scanner.

Immunofluorescence imaging and phenotype quantification
Cells were seeded on fibronectin-coated glass coverslips and treated with the indicated siRNAs, according to the individual experimental protocol as described in 'siRNA transfections'. For analysis, cells were briefly washed with 1 X PHEM buffer (60 mM PIPES, 25 mM HEPES, pH 6.9, 1 mM EGTA, 2 mM MgCl 2 ) and then fixed for 20 min at 23 °C in 4% formaldehyde + 0.5% Triton X-100 (in 1 X PHEM buffer). Following fixation, cells were washed with PBS and incubated 30 min in blocking buffer (3% FBS +0.1% Triton X-100 in PBS). Primary antibodies were incubated for least 1 hr at 23 °C (see Supplementary file 2C for dilutions). Coverslips were then washed with PBS and incubated with the secondary antibodies (Thermo Fisher) for 1 hr at 23 °C. Following a final wash in PBS, coverslips were mounted onto glass microscope slides using Prolong Gold Antifade Reagent with or without DAPI (Thermo Fisher). In the case of KATNA1, localization was confirmed with two independent antibodies (Proteintech -#17560-1-AP; Abcam -ab111881) and two different fixation conditions: -20 °C Methanol for 10 min and PFT (1 x PHEM + 4% PFA +0.5% Triton as described above) (data not shown).
Images were acquired using a Nikon Ti-E inverted microscope equipped with a 60X PlanApo oil immersion objective, an Andor Zyla CMOS camera, and an automated Prior II motorized stage controlled with the Nikon Elements software. For phenotype quantification in Figure 5, the software was used to acquire 49 images using a randomized 7 x 7 grid pattern. The images were then blinded and scored independently by four individuals. For Figure 6, 25 images for each treatment were acquired, blinded, and scored independently by three individuals. Quantification and statistical analyses were performed using GraphPad Prism. Additional files in the H2/H3 groove of the first MIT domain in a Type 1 interaction Skalicky et al., 2012). The MIT domains of SNX15 and SPARTIN both bind exclusively to IST1. The SPARTIN-IST1 interaction had been reported previously Guo and Xu, 2015), and we found that the IST1 MIM 344-366 element provided most of the binding energy ( Figure 3C), in good agreement with previous studies. This Type 3 interaction was visualized in a structure of the SPARTIN MIT-IST1 complex . Ours is the first report of ESCRT-III binding by SNX15, and we found that both IST1 MIM elements contribute significantly, indicating that both elements can simultaneously occupy both the H1/H3 and H2/H3 grooves. SNX15 has been reported to function in endosomal protein trafficking (Phillips et al., 2001), which is also a major site of ESCRT function (see above).
As discussed in the main text, the MIT domains ULK1, VPS9D1 and NRBF2 did not bind ESCRT-III proteins in our screen.